The human pancreatic elastase I gene is transcriptionally silent, despite the apparent integrity of the structural gene. The transcriptional regulatory sequences necessary and sufficient for transcription of the active rat homologue are localized within 205 base pairs (bp) of the transcriptional start and comprise a pancreas-specific transcriptional enhancer of 134 bp immediately upstream of a 71 bp non-specific promoter. The human gene has 58 nucleotide differences within this region, 13 of which are in the three functional elements (A, B and C) that constitute the enhancer. Through cell transfection analyses with a pancreatic acinar tumor cell line, we show that the nucleotide differences in the human 5' flanking gene sequences have inactivated both the enhancer and the promoter. The changes in the three elements of the human enhancer alone are sufficient to inactivate the enhancer; conversely, restoring these to the rat configuration partially restores the activity of the human enhancer. The two mutations in the A element and the four mutations in the B element abolish the binding of the transcription factors previously shown to mediate the activity of these elements. Replacing the active 71 bp rat promoter with the human promoter also prevents expression. Therefore, the evolutionary silencing of the human elastase I gene appears due to mutations that inactivate crucial enhancer and promoter elements.